Yeast extracts have a function of imparting atsumi (thickness), umami, etc. to foodstuffs, and have been widely used as seasonings in the field of foodstuffs. Especially, glutathione (henceforth also referred to as “GSH”), which is a tripeptide consisting of glutamic acid, cysteine and glycine, is known to impart kokumi to foodstuffs (Non-patent documents 1 and 2), and seasonings containing GSH have been developed.
Meanwhile, although the calcium sensing receptor (CaSR), which is a G-protein classified into the class C, has been reported to respond to GSH (Non-patent document 3), the physiological significance thereof has not been clarified. Moreover, this CaSR is present also in the lingual cells, and it was considered to show a certain taste response (Non-patent document 4). Then, it has recently been clarified that this CaSR participates in recognition of kokumi in humans (Non-patent document 5). This reference reported that not only GSH that has been recognized as a kokumi substance, but also several γ-glutamyl compounds similarly respond to CaSR. Furthermore, it has been reported that peptides represented by the general formula γ-Glu-X or γ-Glu-X-Gly (X can represent an amino acid or amino acid derivative other than Cys), for example, γ-Glu-Met, γ-Glu-Thr, γ-Glu-Val-Gly, etc. have a kokumi-imparting effect (Patent document 1). Moreover, the group of esters including S- or O-carboxyalkylated γ-glutamyl or β-asparagyl peptides, and so forth are also reported as kokumi substances (Patent document 2). Although these peptides impart kokumi to foodstuffs like GSH, they do not have a reduced SH group unlike GSH. It is known that a substance having the reduced SH group such as GSH is generally unstable, and titer thereof is reduced with formation of disulfide bond (Patent document 2). However, γ-Glu-X, γ-Glu-X-Gly etc. are considered useful from the viewpoint that the kokumi-imparting peptides not having the reduced SH group are stable.
Tastes sensed after eating change with time, and the tastes are called initial taste, middle taste, and aftertaste in the order from the taste sensed immediately after eating. Although tastes imparted by various substances change with time in various patterns, concerning especially kokumi, a kokumi-imparting substance showing a taste-imparting pattern that imparts strong initial taste is highly desired. It is known that the γ-glutamyl compound, γ-Glu-Abu-Gly, has a kokumi-imparting action that mainly imparts initial-middle taste (Patent documents 3 and 4).
It is known that the synthesis and decomposition of glutathione, which is one of the γ-glutamyl compounds, is catalyzed by several enzymes which make up the γ-glutamyl cycle. In particular, γ-glutamyl transpeptidase is known to transfer the glutamate of GSH at the γ-position to another compound having an amino group, resulting in decomposition of GSH to cysteinylglycine (Non-patent document 6). It is considered that, if the compound having an amino group in this reaction is an amino acid, a dipeptide of γ-Glu-X can be generated as a by-product. However, research about producing these compounds effectively using microorganisms has not been positively performed to date, partially because they are by-products.
An analysis of the fermentation broth of Micrococcus glutamicus can be noted as findings about the dipeptide γ-Glu-X (Non-patent document 7). This reference reported that the fermentation broth was loaded onto various columns to separate peptides and the like, resulting in the isolation of γ-Glu-Glu, γ-Glu-Val, and γ-Glu-Leu. However, these were found as a result of separation with various columns, and the amounts of these peptides contained in the broth were not determined. In addition, there was not reported in all the above examples that γ-Glu-Abu was contained.
GSH is biosynthesized by two kinds of enzymes called γ-glutamylcysteine synthetase, which binds Glu and Cys to generate γ-Glu-Cys, and glutathione synthetase, which binds the generated γ-Glu-Cys and Gly to generate GSH. The substrate specificities of the enzymes were investigated in in vitro enzymatic reactions, and it was reported that γ-Glu-Abu was generated from Glu and Abu as the substrates (Non-patent document 8). However, this report concerns an example using a bacterium, Proteus mirabilis, and does not concern investigation using yeast. Furthermore, although Abu can be used as a substrate in an in vitro enzymatic reaction, any Abu synthetic pathway is not known for yeasts.
Yeast extracts produced from yeast cells as a raw material are seasonings that have been widely used in the field of foodstuffs, and are highly accepted by consumers. Therefore, a yeast extract is more preferred as a carrier of taste substances. Yeast strains containing minerals can be exemplified as the investigation concerning the use of yeast as a carrier of taste substances. It is known that if a metal is added to a medium, yeasts uptake the metal into the cells (Non-patent document 9). In particular, if trace elements such as zinc, iron, copper, manganese, selenium, molybdenum and chromium are added to the medium, yeasts can be used as a supply source of the desired enriched elements as foodstuffs (Patent document 5). From this point of view, methods for producing mineral-containing yeast have been developed (Patent documents 6 to 8).
Furthermore, yeast incorporating such minerals may also enjoy a merit concerning on taste. For example, there can be mentioned the yeast containing a large amount of magnesium (Patent document 9). This reference describes that although magnesium-enriched foodstuffs containing inorganic magnesium salt were also marketed, a strong bitterness and astringency was noted due to the mineral salt. As a result, it was quite more difficult to routinely eat the magnesium-enriched foodstuffs containing inorganic magnesium salt as compared to foodstuffs containing naturally occurring magnesium. In that context, Patent document 9 discloses a method to produce a foodstuff containing magnesium in natural form by letting yeast uptake magnesium. As for nutritional merit of yeast that uptakes minerals, the technique disclosed in Patent document 10 can be exemplified. According to this reference, although zinc contributes to improvement of taste disorder and generative function, etc., zinc is still not taken in sufficient amounts. If zinc is added during the yeast cultivation process, yeast uptakes zinc into cells. In this case zinc is not accumulated in the cells as water-soluble form, but zinc is highly accumulated in the cells as amorphous zinc form which binds with a protein or an amino acid. When the amorphous zinc is taken into the human body, the amorphous zinc is more efficiently absorbed into the body compared with crystalline zinc. As a result, absorption of zinc into the body can be improved by taking zinc-containing yeast, as compared to simply taking zinc itself.
As described above, a method comprised by making yeast uptake a target substance and adding either the yeast or a yeast extract to foodstuffs can enjoy various advantages as compared to simply adding the target substance itself to foodstuffs. However, unlike minerals, which are essential nutrients, the ability of yeast to uptake an amino acid or a peptide is strictly controlled, and simply applying the technique for incorporating minerals into yeast to the techniques for uptake of amino acid or peptides was considered to be difficult.
As described above, although yeast cells or yeast extracts are preferred as a carrier of a γ-glutamyl compound such as γ-Glu-X as a kokumi-imparting agent, there have been substantially no reports about yeast cells containing such a γ-glutamyl compound, and a method for producing an extract prepared from the cells.